STUDIES ON MECHANISM OF MITOSIS
STUDIES ON MECHANISM OF MITOSIS
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DOI：
10.1111/j.1749-6632.1975.tb19217.x
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发表时间：
1975-01-01
影响因子：
5.2
通讯作者：
VANDERSLICE, K
中科院分区：
文献类型：
--
作者：
MCINTOSH, JR;CANDE, Z;VANDERSLICE, K
材料和方法用标准技术培养哺乳动物细胞（HeLa、WI-38、CHO和PtK）。对于光学显微镜，将细胞在玻璃盖片上传代培养，所述玻璃盖片可以倒置到玻璃载片上（使用石蜡膜的薄片作为间隔物）并上蜡以形成将支持细胞分裂的开始和完成约4小时的简单室。对于电子显微镜，细胞固定后，布林克利等的方法。O或在4%戊二醛、4%20 M Carbowax@（Cx）的0.1 M哌嗪-N-N ′-（2-乙磺酸）混合物中，用NaOH（PIPES缓冲液）调节至pH 6.9。这种固定剂必须在使用前配制新鲜。将固定的细胞在缓冲液中冲洗，用OsO处理，脱水，并通过标准技术包埋。用相位显微镜从塑料晶片中选择单细胞，切下，并以选定的方向连续切片用于电子显微镜。对于高压电子显微镜中的显微镜检查，将细胞固定并如所述包埋，并以1 μ m的标称厚度切片。在碳和Formvafl涂覆的狭缝网格上拾取约10个切片，在室温下用乙酸双氧铀和柠檬酸铅各自染色约30分钟。
MATERIALS AND METHODSMammalian cells (HeLa, WI-38, CHO, and PtK,) were cultured by standard techniques. For light microscopy, the cells were subcultured on glass cover slips which can be inverted onto a glass slide (using slivers of parafilm as spacers) and waxed down to form a simple chamber that will support the initiation and completion of cell division for about 4 hours. For electron microscopy, the cells were fixed after the method of Brinkley et aI.,? O or in a mixture of 4% glutaraldehyde, 4% 20 M Carbowax@(Cx) in 0.1 M piperazine-N-N’-(2-ethane sulfonic acid) adjusted to pH 6.9 with NaOH (PIPES buffer). This fixative must be made up fresh before use. Fixed cells were rinsed in buffer, treated with OsO,, dehydrated, and embedded by standard techniques. Single cells were selected from wafers of plastic with a phase microscope, excised, and serially sectioned in a chosen orientation for electron microscopy. For microscopy in a high-voltage electron microscope, the cells were fixed and embedded as described and sectioned at a nominal thickness of 1 pm. About 10 sections were picked up on a carbon and Formvafl-coated slot grid, stained with uranyl acetate and lead citrate for about 30 min each at room